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PubMed1985

Synthesis and degradation of type I procollagen mRNAs in cultured human skin fibroblasts and the effect of cortisol.

The metabolism of type I procollagen mRNAs was studied in confluent cultures of human skin fibroblasts using the recombinant plasmids Hf677 and Hf32, which contain DNA sequences complementary to human pro-alpha 1(I) and pro-alpha 2(I) mRNAs, respectively. The ratio for the amount of pro-alpha 1(I) mRNAs to pro-alpha 2(I) mRNAs was 2.06:1. These mRNAs were labeled during in vitro transcription of isolated nuclei in the ratio 2.16:1 and during a pulse in cellulo in the ratio 1.96:1, suggesting that the two species are synthesized in the ratio 2:1. No significant differences were found in the rates of degradation, the half-lives of the pro-alpha 1(I) and pro-alpha 2(I) mRNAs being 9.2 and 8.4 h, respectively. Addition of 10 microM cortisol to the growth medium reduced the amounts of the type I procollagen mRNAs by half in 6 h, leaving their ratio unaltered. Incubation of the cells with cortisol had only minor effects on the subsequent synthesis of the mRNAs in isolated nuclei in vitro, whereas the degradation rates in cellulo were distinctly accelerated.

Cells, Cultured↗
PubMed1977

Kinetics for the secretion of procollagen by freshly isolated tendon cells.

Fibroblasts isolated by enzymic digestion of chick embryo tendons were incubated for several hours in suspension under conditions in which they were in steady state in terms of the synthesis and secretion of procollagen. Under these conditions, the cells synthesized and secreted about 630 microgram of procollagen/10(9) cells/h. The cells were labeled with [14C]proline for 15 to 120 min and then the kinetics of secretion were followed by chasing the label and assaying the 14C-peptides digestible by collagenase in the cells and in the medium. The results demonstrated that secretion of collagenase-digestible peptides did not follow the kinetics of a single first order process but suggested at least two pseudo-first order process with half-times of 14 and 115 min. The [14C]procollagen secreted during 0 to 30 min and 90 to 120 min of chase was the same in terms of the ratio of pro-alpha1 to pro-alpha2 chains, the size of the pro-alpha chains, the extent of interchain disulfide bonding, the extent of prolyl hydroxylation, and the degree of helicity as tested by resistance to pepsin digestion. Addition of ascorbic acid to the incubation medium increased slightly the extent of prolyl hydroxylation but did not alter the kinetics of secretion. The results suggested that the kinetics of secretion are influenced by a two-compartment system in which at least one metabolic pool contributing to the secretory process is present as a "side pocket."

Animals↗
PubMed1981

[Radioimmunoassay for type II amino-terminal procollagen peptide. Technical note].

We have studied the immunoreactivity of human sera and synovial fluids in a radioimmunoassay which utilizes an anti-bovine procollagen type III antiserum and the bovine type III procollagen peptide as standard and tracer. We have noticed a complete cross reaction between bovine col 1-3 and human synovial fluid, which thus contains substance (s) having the same antigenic determinant (s) as col 1-3. On the contrary, we have demonstrated non parallelism between col 1-3 and human serum, whether the serum was obtained from normal subjects or patients suffering from various diseases. These results suggest that immunoreactive substances related to col 1-3 and detected in sera are at least partially different from those detected in synovial fluids.

Animals↗
PubMed1978

Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen.

Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.

Animals↗
PubMed1984

[Development of polycythemia to myelofibrosis. Monitoring by the assay of the procollagen III amino-propeptide].

The conventional methods used in the follow-up of myelofibrosis being both invasive and expensive, we propose an easy and sensitive immunoassay of the procollagen III aminoterminal peptide (propeptide) which is separated and released in serum during synthesis of collagen III by bone marrow fibroblasts. This propeptide was assayed in the sera of 25 healthy adults (controls), 48 patients with polycythaemia vera and 20 patients with myelofibrosis (secondary to polycythaemia in 14). Significant differences in mean values were found between controls (7.6 +/- 2.1 ng/ml), patients with polycythaemia (9.9 +/- 4.3 ng/ml) and patients with myelofibrosis (38.1 ng/ml). There was some overlapping between values in controls and in patients with polycythaemia, partly due to the fact that 12 of these 48 patients without reticulin bone marrow fibrosis had normal serum propeptide levels. Propeptide values in patients with myelofibrosis were much scattered (range: 13-103 ng/ml). Moreover, values above 25 ng/ml were associated with myelofibrosis of recent onset (less than or equal to 2 years) and values below 25 ng/ml with myelofibrosis of more than 4 years' duration. The procollagen III aminoterminal peptide immunoassay therefore is a non-invasive and sensitive method for accurate assessment of bone marrow progressive involvement in myeloproliferative diseases associated with myelofibrosis. It could also be used to evaluate the effectiveness of antifibrosing agents.

Adult↗
PubMed1981

Propeptides of procollagen V (A,B) in chick embryo crop.

The biosynthesis of type V (A,B) collagens was recently found to proceed through the sequential forms pro alpha (A,B), p alpha (A,B), and f alpha (A,B). All these chains are larger than the A,B chains extracted from tissues after pepsin digestion. This report shows that all forms contain substantial peptides which are resistant to bacterial collagenase and concludes that type V collagens differ from the intestitial collagens (types I, II, III) in retaining large noncollagenous peptides in tissues. A peptide becomes transiently attached to the processing intermediate p alpha A by a reducible linkage. The conversion of procollagen V to p-collagen V was inhibited by colchicine and arginine. Previously, the disulfide-linked heterotrimer [(pro alpha B)2 (pro alpha A)] was found and additional procollagens containing only B type molecules were inferred. Further investigations reported here agree with these conclusions and also indicate that some trimeric molecules containing more than one chain related to A may exist.

Amino Acids↗
PubMed1978

Partial characterization of human collagen and procollagen secreted by human corneal stromal fibroblasts in cell culture.

Human corneal stromal fibroblasts were established into cell culture. Fractions highly enriched for collagen and procollagen were purified from the medium and cell layer. Analysis using polyacrylamide gel electrophoresis and DEAE cellulose chromatography indicated the presence of a heterogeneous population of procollagen molecules ranging in molecular weights from 200,000 to 120,000 daltons along with alpha1 and alpha2 collagen chains. Cyanogen bromide peptides of a corneal peptide fraction migrated concomitantly with type I standard cyanogen bromide peptides.

Cells, Cultured↗
PubMed1983

The molecular defect in a nonlethal variant of osteogenesis imperfecta. Synthesis of pro-alpha 2(I) chains which are not incorporated into trimers of type I procollagen.

Cultured fibroblasts were examined from a patient with a nonlethal form of osteogenesis imperfecta. As reported previously (Nicholls, A. C., Pope, F. M., and Schloon, H. (1979) Lancet 1, 1193), the cells synthesized and secreted a type I procollagen which lacked pro-alpha 2(I) chains and consisted of a trimer of pro-alpha 1(I) chains. No pro-alpha 2(I) chains were recovered from the medium under conditions in which nonhelical pro-alpha 1(I) and pro-alpha 2(I) chains were readily detected in the medium of normal fibroblasts incubated with the hydroxylase inhibitor, alpha, alpha'-dipyridyl. Examination of cellular proteins demonstrated that the fibroblasts synthesized both pro-alpha 1(I) and pro-alpha 2(I) chains. The cellular pro-alpha 2(I) chains did not, however, become disulfide-linked into dimers or trimers of pro-alpha chains. Since the association of pro-alpha chains during the biosynthesis of type I procollagen is directed by the conformation of the COOH-terminal propeptides, the data suggest that the pro-alpha 2(I) chains synthesized by the fibroblasts have a mutated structure in the COOH-terminal propeptides which markedly reduces their affinity for pro-alpha 1(I) chains. A further observation was that the ratio of newly synthesized pro-alpha (I):pro-alpha 2(I) chains in the patient's fibroblasts was 7.18 +/- 0.58 S.E. instead of the value of 2.25 +/- 0.16 S.E. seen in control fibroblasts. One possible explanation for the high ratio is that the proband is homozygous for a mutation altering the structure of the pro-alpha 2(I) chain and that a secondary effect of the structural mutation is a decreased rate of synthesis of pro-alpha 2(I) chains.

2,2'-Dipyridyl↗
PubMed1983

Lavage type III procollagen N-terminal peptides in human pulmonary fibrosis and sarcoidosis.

Levels of type III procollagen N-terminal peptide were immunoassayed in sera and bronchoalveolar lavage fluids from healthy volunteers and patients with idiopathic pulmonary fibrosis or sarcoidosis. Peptide levels normalized to protein were higher than corresponding serum levels in all groups, suggesting that the lung is a producer of this peptide. Levels were significantly elevated in both patient groups, suggesting increased rates of type III collagen synthesis. Values were highest in idiopathic pulmonary fibrosis samples. Correlation of peptide levels with clinical severity of disease and lavage cell profiles was poor, perhaps because the latter reflects aspects of the disease process other than that stage in which type III collagen synthesis is altered. Lavage analyses of procollagen peptides may provide an important index of altered collagen synthesis in interstitial lung disease.

Amino Acid Sequence↗
PubMed1982

Inhibition of protein synthesis in vitro by procollagen-derived fragments is associated with changes in protein phosphorylation.

The NH2-terminal extension fragment (Col 1) of the pro alpha 1(I) procollagen chain selectively inhibits the translation of procollagen mRNA in a reticulocyte lysate system, whereas the reduced and alkylated fragment (AE-Col 1) and its proteolytically derived peptides inhibit the translation of all mRNAs (Hörlein, D., McPherson, J., Goh, S. H., and Bornstein, P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 6163-6167). The latter inhibitory function, which occurs at the level of polypeptide chain initiation, has now been shown to be associated with an increase in phosphorylation of an Mr = 94,000 protein. The time span required for observation of changes in phosphorylation and in inhibition of protein synthesis is similar. Since AE-Col 1 can serve as a substrate for casein kinase II, we suggest that phosphorylation of AE-Col 1 and its derivatives may be required for their activity.

Animals↗
PubMed1994

Identification of integrin alpha 2 beta 1 as cell surface receptor for the carboxyl-terminal propeptide of type I procollagen.

The carboxyl-terminal propeptide of procollagen type I (CPP-I) plays a key role in the regulation of collagen fibrillogenesis. In addition, it has been reported that, after cleavage from procollagen, CPP-I exerts feedback control of collagen biosynthesis. To further elucidate the mechanisms involved in each of these processes, we have investigated the nature of cell surface receptors for CPP-I. CPP-I affinity chromatography, using detergent extracts of iodinated HT1080 cells and EDTA elution, resulted in the isolation of two polypeptides of molecular mass 160 and 110 kDa. Since the migratory behavior of these polypeptides under nonreducing and reducing conditions was characteristic of a subset of integrin receptors, their reactivity with anti-integrin monoclonal antibodies was tested. Antibodies directed against the alpha 2 and beta 1 subunits specifically immunoprecipitated both CPP-I-binding polypeptides, indicating that the CPP-I receptor is the integrin alpha 2 beta 1. CPP-I was found to support the attachment and spreading of HT1080 cells, demonstrating that it can function as an adhesion protein. Two other approaches supported the identification of alpha 2 beta 1 as the CPP-I receptor. First, anti-functional anti-integrin monoclonal antibodies directed against the alpha 2 and beta 1 subunits completely abrogated the adhesive activity of CPP-I and, second, highly purified CPP-I bound specifically to alpha 2 beta 1-containing integrin preparations in a solid-phase receptor-ligand binding assay. These findings have important implications for the function of fibrillar collagen carboxyl-terminal propeptides and for the role played by integrins in the regulation of cellular phenotype.

Antigens, CD↗
PubMed1979

Biochemical characterization of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture.

Collagen synthesis was studied in monolayer cultures of rabbit corneal endothelial cells by following [14C]proline and [3H]glucosamine or [3H]fucose incorporation into a fraction enriched for collagen and its precursor molecules. Sodium dodecyl sulfate gel electrophoresis of this fraction showed that it consisted of a high-molecular-weight (greater than 300,000 daltons) polypeptide. This component was collagenase sensitive and, in the presence of dithiothreitol, gave rise to two polypeptides of the apparent molecular weights of 200,000 and 160,000 daltons. Pepsin digestion of this material destroyed all the high-molecular-weight material and gave rise to a single collagenase-sensitive component of an apparent molecular weight of 115,000 daltons. This 115,000 dalton material is similar to previously observed basement membrane collagens, and the 160,000 and 200,000 dalton components are probably precursor chains of basement membrane collagen. The very-high-molecular-weight material (greater than 300,000 daltons) may represent a disulfide-linked complex of these precursor chains. DEAE-cellulose column chromatography confirmed the presence of a single procollagen species distinct from the collagen fraction. Amino acid analysis of collagen and procollagen fractions showed a decreased hydroxyproline value as compared with previously reported basement membrane collagens or collagen precursors.

Amino Acids↗
PubMed1995

[Effects of alveolar macrophage conditioned media from interstitial lung disease patients on the procollagen mRNA expression in human lung fibroblasts].

We investigated the effects of alveolar macrophage (Am) conditioned media from interstitial lung disease (ILD) patients on lung fibroblast proliferation and procollagen mRNA expression in cells. 13 patients with ILD and 8 patients with bronchial carcinoma (BC) received bronchoalveolar lavage (BAL). The BAL fluid was collected and centrifuged to obtain cells of two groups. The cells were washed and incubated at 37 degrees C and 5% CO2 for 2 hours in DMEM with 10% FCS. Then the adherent Am was added with DMEM with 0.1% FCS. (2ml/10(6) cells), and cultured at 37 degrees C and 5% CO2 for 24 hours. The supernatant was obtained by centrifugation and stored in -20 degrees C for experiment. Lung fibroblasts were planted in 96 well microtitle plate and cultured to confluence. Each well was added 100 microliters Am conditioned medium and incubated for 16 hours, then added 5 uci 3H-TdR for measuring the fibroblast proliferation and incubated till 24 hours. The cells were collected and counted. The DMEM with 0.1% FCS was used as control. When stimulated with Am conditioned media from ILD patients, fibroblast proliferation increased 71%, but for media from BC patients, it just increased 14% (P < 0.05). After stimulated with conditioned media for 24 hours, the cells were trypzied, collected, ashed and isolated total RNA with guanine method. Several samples' total RNA was subjected to electrophoresis in 1% agaroseformaldehyde gel to test specificity of procollagen al (I) and al (III) cDNA probes. 10 micrograms total RNA of each sample was bloted to nitrit membrane and baked at 80 degrees C for 2 hours.

Cell Division↗
PubMed1994

Persistence, re-expression, and induction of pulmonary arterial fibronectin, tropoelastin, and type I procollagen mRNA expression in neonatal hypoxic pulmonary hypertension.

Changes in the structure and function of muscular pulmonary arteries are crucial for normal adaptation to extrauterine life and rapid changes in matrix protein gene expression are likely necessary for this adaptation. We hypothesized that pathological stimuli imposed at the time of birth would alter developmental changes in matrix protein gene expression thereby affecting the normal post-uterine changes in pulmonary hemodynamics. We used in situ hybridization to examine the normal developmental expression of three extracellular matrix protein mRNAs, fibronectin, tropoelastin, and alpha 1 (I) procollagen, in muscular pulmonary arteries of both fetal and neonatal calves and assessed the impact of severe hypoxia-induced pulmonary hypertension on their expression. Morphometric techniques were used to assess whether changes in matrix protein mRNA levels were related to changes in matrix fiber accumulation. Exposure to chronic hypoxia postnatally resulted in the persistence, reexpression, and induction of fibronectin, tropoelastin, and alpha 1 (I) procollagen mRNAs, respectively, in muscular pulmonary arteries. In each case the hybridization signal was localized primarily to the adventitial layer of the vessel. Morphometric analysis showed that the increased hybridization signals seen correlated with an increase in both vascular elastin and collagen fiber volumes in the adventitial layer. We conclude that the change in expression of matrix genes in the pulmonary artery wall during exposure to chronic hypoxia is an important adaptive response to changes in hemodynamic factors and/or oxygen tension. The unchecked increase in matrix protein expression seen likely contributes to the pathological pulmonary arterial structural remodeling and loss of vasoreactivity that occurs during the development of severe neonatal pulmonary hypertension.

Animals↗
PubMed1994

Familial high serum concentrations of the carboxyl-terminal propeptide of type I procollagen.

We describe a family with an apparently autosomal-dominant trait that caused extremely high circulating concentrations of the carboxyl-terminal propeptide of type I procollagen (PICP). All family members examined had normal values for other biochemical markers of bone formation and degradation and no related clinical abnormalities. Furthermore, their serum concentrations of the amino-terminal propeptide of type I procollagen (PINP) were normal. Although PINP and PICP are released from the same precursor molecule, PINP is cleared from the circulation via the scavenger receptor in liver endothelial cells, whereas PICP is cleared via the mannose receptor of these cells. We thus hypothesize that the clearance of circulating PICP is compromised in the affected subjects of this family, the result of either a defective mannose receptor function or an abnormal molecular structure of their PICP.

Adolescent↗
PubMed1993

Enhanced expression of mRNA for transforming growth factor-beta, type I and type III procollagen in human post-burn hypertrophic scar tissues.

To explore the possible role of locally synthesized transforming growth factor beta-1 (TGF-beta 1) and procollagen gene expression in postburn hypertrophic scars, we have compared mRNA levels for type I and type III procollagen and TGF-beta 1 in human hypertrophic scar tissue with normal dermis obtained from the same patients as a control. Northern blot analysis of total RNA extracted from hypertrophic scar tissue and normal skin demonstrated two transcripts for the pro-alpha 1(I) chain (5.8 kb and 4.8 kb) and for the pro-alpha 1(III) chain (5.4 kb and 4.8 kb) and one transcript (4.9 kb) for TGF-beta 1. Quantitative analysis of dot blot autoradiograms of mRNA from three samples of hypertrophic scar tissue and normal skin showed average increases of 102% (p < 0.05) for pro-alpha 1(I), 91% (p < 0.06) for pro-alpha 1(III), and 61% (p < 0.05) for TGF-beta 1. Three additional hypertrophic scar samples were quantitatively analyzed on Northern blots and showed increases of 246%, 102%, and 250% of the specific messages for pro-alpha 1(I), pro-alpha 1(III), and TGF-beta 1 relative to a normal skin control. Two transcripts (4.9 kb and 2.5 kb) for TGF-beta 1 were identified in cultured fibroblasts. In contrast to the results from tissue, the level of these transcripts in fibroblasts cultured from hypertrophic scar tissue and normal skin were not significantly different, suggesting that the synthesis of this growth factor is stimulated in tissue by a presently unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Deletion of a large domain in recombinant human procollagen II does not alter the thermal stability of the triple helix.

A construct of the human gene for procollagen II (COL2A1) was prepared with an internal deletion of 5 kilobases that removed 12 exons coding for 291 amino acids from near the NH2 terminus of the triple helix. The construct was then used to transfect stably a human tumor cell line (HT-1080), and clones secreting internally deleted pro alpha 1(II) chain of procollagen II were isolated. The protein was purified, and the thermal stability of the triple-helical domain was assayed by brief protease digestion. The thermal stability of the internally deleted protein was the same as that of intact collagen II even though the triple helix was 39% shorter. Additionally, the thermal stability of the collagenase A fragment was the same as that of the collagenase A fragment of normal collagen II even though it was 38% shorter. Analysis of the results suggested that the thermal stabilities of large fragments of collagen II depended primarily on their contents of -Gly-Pro-Hyp-triplets corrected for length.

Amino Acid Sequence↗
PubMed1996

[Procollagen type I C-propeptide in kidney transplant recipients].

The excessive production of parathormone may persist or return after renal transplantation, so that latent hyperparathyroidism in time may lead to loss of bone mass and of the new kidney; a precocious diagnosis of this disease represents the best preventive measure. On a group of 50 successfully undergone kidney transplant patients (group A: 38 immunosuppression therapy with corticosteroids, azathioprine and cyclosporine A patients; group B: 12 immunosuppression therapy with azathioprine and ciclosporine A patients) we have evaluated also the most common markers of hyperparathyroidism, C-propeptide of procollagen of type I (PICP), that is a product of procollagen degradation and it represents in the serum a direct measure of osteoblastic bone activity. Our results showed alkaline phosphate, osteocalcin and PICP increase, which are index of osteoblastic activity and urinary collagen cross-links pyridinoline and deoxypyridinoline increase which are index of bone osteoclastic activity. The survey revealed a linear significant correlation only between PICP and pyridinoline and cross link deoxypyridinoline (p < 0.05). In this group of patients the only PICP could not have a diagnostic meaning. Owing to the particularly bone metabolism of our patients, that probably feel the effects of hyperparathyroidism of the pre-transplant period, PICP turns out insufficient to study the persistent or returned hyperparathyroidism.

Adult↗